The objective of this project is to define the initial, intracellular events of glucocorticoid hormone action and steroid hormone action in general. Such studies are of particular current relevance since steroid hormone receptors are arguably the best understood regulators of eukaryotic gene transcription. The first step of steroid binding to the intracellular receptor is followed by activation of the receptor-steroid complex to a DNA/nuclear-binding species that then associates with those nuclear acceptor sites involved in the regulation of transcription of selected genes in specific cells. We have found that the steroid binding domain of the glucocorticoid receptor not only binds steroid but also is sufficient for the association of a non-receptor protein, heat shock protein 90, which is required for steroid binding and whole cell localization of receptors. This result will greatly aid future molecular studies of the initial steps in steroid hormone action. The nuclear binding of activated complexes is required for the expression of steroid responses and is different for glucocorticoid and antiglucocorticoid steroids. We have found that this binding cannot be reproduced in vitro using methodologies that work for other macromolecules. This suggests that novel processes are involved in glucocorticoid receptor binding to nuclei. Efforts to define these new processes, and thus permit future in vitro studies of nuclear binding, should be facilitated by the use of aurintricarboxylic acid, which we found readily distinguishes between receptors bound to DNA and non-DNA acceptor sites. In vivo nuclear-bound activated receptor-steroid complexes often modulate the transcription of selected proteins in a tissue specific manner. Our finding of a new tissue selective element in the expression of a model glucocorticoid inducible gene represents a further advance towards identifying all of the components required for cell-free studies of tissue specific gene expression. Collectively, our current findings contribute to our long term goal of defining the mechanisms of steroid hormone action at a molecular level.